Maize Single Tetrad-stage Microspore Sequencing

Abstract

This protocol records a method 1) to isolate single microspores from the same tetrad, 2) to lyze cell, then 3) to amplify the single cell DNA, and finaly 4) to prepare library for Illumina sequencing. The method development is based on Maize tetrad-stage microspores. Please modify Isolation steps appropriately, If your experimantal materials are not Maize.

Reagents

D-sorbitol,

QIAGEN REPLI-g Single Cell Kit,

10×PCR Buffer (Takara),

dNTP (Katara),

rTaq (Katara),

d2H2O,

2% Agarose gel

TruSeq DNA Sample Prep v2 Kit (Illumina, San Diego, US)

Agencourt Ampure XP beads (Beckman Coulter)

80% EtOH

MinElute Gel Extraction Kit (QIAGEN, Hilden, Germany)

Equipment

Programmable Microinjector PM 2000 (MDI, South Plainfield, USA),

PMP-102 Micropipette Puller (MDI, South Plainfield, USA),

4×microscopy,

Bioruptor UCD-200, Seraing, Belgium

Universal PCR cycler

Procedure

Isolate single microspores from a single tetrad

In the field, select plants presenting tetrad-stage pollen, and harvest the top ~30 cm part containing tassels into a flask filled with water.

Note 1: Under these conditions, the immature pollens would keep developing, so that fresh tetrad-stage cells can be provides from upper to lower spikelets continuously at intervals of ~30min.

In a spikelet there are three older and three younger anthers. Release cells from the three same-stage anthers into Isolation Buffer on a slide. Identify samples containing the last-stage tetrads, from which are easy to separate into microspores and do not adhere to the glass tools.

Note 2: F1 tetrads have a high internal osmotic pressure. If micro-manipulating F1 tetrads, Isolation Buffer should be 27% D-sorbitol solution to balance the high pressure; if micro-manipulating tetrads from Inbred lines, much lower concentration (i.e. physiological concentration), of osmoticum is also sufficient. Do not use normal saline (NS) as Isolation Buffer, because metal ions may interfere with the amplification reaction later. From step 2) to step 5), cells should be kept in the Isolation Buffer.

Isolate a single tetrad into a new drop of Isolation Buffer using a thin glass pipette.

Note 3: Use a thin glass pipette system and Programmable Microinjector PM 2000 (MDI, South Plainfield, USA) to isolate single tetrads (step 3)) and single microspores (step 5)), and to destroy the cell wall (step 4)), under 4×microscopy.

Repeated aspiration in and out was used to destroy the cell walls and disrupt the tetrads. Then, four microspores can be separated without cell wall.

One by one, place cells in less than 0.5μl Isolation Buffer into PCR wells filled with 4μl PBS (QIAGEN, Hilden, Germany). The PCR wells should be kept on ice.

Note 4: When aspirating a microspore in the glass pipette, make sure it is still visible under the microscope. After blowing it to a PCR well, the cell should not be seen in the pipette any more, to ensure that the cell is now in the PCR well for the next reaction.

Note 5: Tips should not touch the surface of the sample inside PCR well when adding reagents. Do not shake the PCR well for mixing, to avoid losing the single cell sample and reducing the integrity of the DNA.

Note 6: Step 6) to step 7) are based on standard protocol of QIAGEN REPLI-g Single Cell Kit.

MDA Amplification of single cell DNA

Prepare the following mixture: 29μl Reaction Buffer, 9μl sc d2H2O and 2μl reaction enzyme per one reaction. Add the mixture to PCR well containing 10μl sample. Centrifuge briefly and incubate at 30℃ for 8 h. Finally, incubate at 65℃ for 3min. Store it at -20℃ if not proceeding to the next step immediately.

Note 7: Similar to note 5, tips can’t touch the surface of the sample inside PCR well when adding reagents. Do not shake the PCR well for mixing. These precautions avoid reducing the Integrity of DNA.

Note 8: MDA products are suitable for Single Nucleotide Variants (SNV) analysis, but not appropriate for quantification analysis (Copy Number Variations, CNV).

Quality control of whole-genome amplification products:

Dilute the amplification product 1:50. Use the 10 markers (Table 1) to amplify the diluted DNA by PCR.

Detect the PCR product by 2% Agarose gel electrophoresis. Sample with no less than 8 of the 10 markers successfully amplified, is considered to be of a quality sufficient to make a library.

Measuring DNA concentration of amplification product

Add 4μl amplification product into 100μl d2H2O. Measure the DNA concentration by Nanodrop 2000. The concentration should be approximately 40~50 ng/μl.

DNA Library construction

Dilute DNA to 1.2μg in 60μl. Then fragment it by sonication (Bioruptor UCD-200, Seraing, Belgium) at ~5℃for a few cycles (30s sonication and 30s rest per one cycle), so that most of the DNA fragments range from 300bp to 500bp.

Note 9: Detection of fragments’ size is by 2% Agarose gel electrophoresis; if most of the fragment are still larger than 500bp, continue sonicating a few more cycles until the suitable size of fragments are generated. The number of cycles depends on when the fragments reach 300bp to 500bp; the mean number of cycles would be ten.

End repair: Add 40μl End Repair Mix and 10μl Resuspension Buffer to 50μl of DNA fragmented. After mix thoroughly, incubate it at 30℃ for 30min.

Purification #1st by Agencourt Ampure XP beads (Beckman Coulter, cat. no. A 63881): Keep the Beads at room temperature (RT) and vortex briefly. Add 160μl Beads to 100μl of the end repaired sample. Mix thoroughly, then incubate at RT for 15min. Place this sample on a magnetic stand at RT for 15min. Aspirate and discard the supernatant using a 200μl pipette. Add 200μl fresh 80% EtOH, incubate at RT for 30s, remove and discard the supernatant. Add 200μl fresh 80% EtOH again to complete the washing, incubate at RT for 30s, remove and discard the supernatant. Let the sample dry at RT for ~5-10min. Remove the sample containing Beads from the magnetic stand, then add 17.5μl Resuspension Buffer to the Beads. Mix thoroughly and incubate at RT for 2min. Place the sample again on the magnetic stand at RT for 5min. Finally, transfer 15μl of the supernatant into a new PCR well for the next step.

Adenylate 3’ end: Add 2.5μl Resuspension Buffer and 12.5μl A Tailing Mix to the PCR well containing 15μl of sample purified as described above. Mix thoroughly, and incubate at 37℃ for 30min.

Purification #2nd using Agencourt Ampure XP beads (Beckman Coulter, cat. no. A 63881): Place the Beads at room temperature (RT) and vortex. Add 42.5μl Beads to 42.5μl of the Ligation sample. Mix thoroughly, then incubate at RT for 15min. Place the sample on a magnetic stand at RT for 5min. Remove and discard the supernatant using a 200μl pipette. Add 200μl fresh 80% EtOH, incubate at RT for 30s, remove and discard the supernatant. Add 200μl fresh 80% EtOH again to complete the washing, incubate at RT for 30s, remove and discard the supernatant. Let the sample dry at RT for ~5-10min. Keep this sample containing Beads away from the magnetic stand, then add 22.5μl Resuspension Buffer. Mix thoroughly and incubate at RT for 2min. Place the sample on the magnetic stand at RT for 5min. Finally, transfer 20μl of the supernatant into a new PCR well for the next step.

Size selection by MinElute Gel Extraction Kit (QIAGEN, Hilden, Germany, cat. no. 28604): After 2% Agarose gel electrophoresis, excise gel segment containing the main target band, which should be limited in size range of 100bp. Add 900μl QG Buffer to 2ml tube containing 300mg gel. Incubate it at RT until gel melts completely. Add 300μl isopropanol, and mix thoroughly. Place a MinElute column in a new 2ml collection tube. Transfer 750μl of the melted sample in the MinElute column, centrifuge for 1min at 13000 rpm and discard the flow-through. Repeatedly transfer another 750μl and centrifuge. Add 500μl QG Buffer to the column, centrifuge for 1min and discard flow-through. Add 750μl PE Buffer to the column, stand for 5min, then centrifuge for 1min and discard flow-through. Centrifuge for 1min one more time to remove residual ethanol completely. Place the column into a clean 1.5ml tube. Let it dry at RT for 5min. Add 23μl EB Buffer onto the center of column membrane, incubate it at RT for 3min, then centrifuge it for 1 min. The flow-through is collected and added onto the center of the same column membrane, incubate it at RT for 3min, then centrifuge for 1 min. Now the flow-thorough is the sample purified for PCR reaction.

PCR amplification of library: Add 5μl Primer and 25μl PCR Mix to 20μl of the Purified sample above. Mix thoroughly, and perform PCR with the following cycling parameters:

Process Temperature Duration Cycles see Table 3.

Purification #3rd by Agencourt Ampure XP beads (Beckman Coulter, cat. no. A 63881): Keep the Beads at room temperature (RT) and vortex. Add 50μl Beads to 50μl of the PCR sample. Mix thoroughly, then incubate it at RT for 15min. Place this PCR sample on a magnetic stand at RT for 5min. Remove and discard the supernatant using a 200μl pipette. Add 200μl fresh 80% EtOH, incubate at RT for 30s, remove and discard the supernatant. Add 200μl fresh 80% EtOH again to complete the washing, incubate at RT for 30s, remove and discard the supernatant. Let the sample dry at RT for ~5-10min. Place the sample with the Beads away from the magnetic stand, then add 22.5μl Resuspension Buffer to the Beads. Mix thoroughly and incubate at RT for 2min. Place the sample on the magnetic stand at RT for 5min again. Finally, transfer 20μl of the supernatant into a new PCR well; it is the library. Store it at -20℃, up to two months. Go on to qualify and quantify it for pair-end sequencing on Illumina Hiseq 2000 platform.